The antigen-antibody reaction is the basis for all immunological test methods. Special proteins called antibodies are produced by mammals in response to the presence of an antigen, that is a foreign substance, usually a protein. This normal body response to a foreign protein had led to the development of a number of techniques which are used to diagnose various mammalian diseases, disorders and physiological conditions, such as pregnancy, which is neither a disease nor a disorder. In vitro tests for the presence of a suspected protein, antigen or antibody in a biological sample are carried out by adding the immunological counterpart to the biological sample, i.e., add antigen if the test is for the presence of antibody or add antibody if the test is for the presence of antigen. If the suspected protein is present, and resulting antigen-antibody reaction can be demonstrated by precipitation or agglutination of the antigen-antibody complex. This antigen-antibody reaction is usually very difficult to detect visually. For this reason either antibodies or antigens are bound to latex polymer particles so that when agglutination involving the antigen or, alternatively, the antibody bound to the latex particles, does occur it can be detected visually. Agglutination is characterized by the clumping of the latex polymer particles from an otherwise smooth suspension.
In a direct latex agglutination test for the detection of the presence of an antigen in a biological sample, the biological sample is mixed with a suspension containing antibodies of said antigen bound to latex polymer particles. If antigen is present in the sample it will react with the antibodies to form an agglutant also referred to as clumps or precipitant. If no antigen is present in the sample the mixture will keep its appearance as a smooth suspension. A direct latex agglutination test for the detection of an antibody in a biological sample works on similar principles whereby antigen of said antibody are bound to latex particles.
In an indirect latex agglutination test for the detection of the presence of an antigen in a biological sample, the sample is mixed with antibodies of said antigen and allowed to incubate. If antigens are present in the biological sample they will inactivate (form a complex with) the antibodies so that when a latex-antigen suspension is subsequently added to the reaction mixture no agglutination will occur and the test mixture retains a smooth suspension appearance indicating a positive response. If no antigens are present in the biological sample then antibodies are available to react with a latex-antigen suspension when it is introduced into the reaction mixture resulting in agglutination indicating a negative result. An indirect latex agglutination test for the detection of the presence of antibodies in a biological sample works on similar principles whereby the sample is initially mixed with antigens and thereafter mixed with antibodies bound to latex particles.
Some commercial examples of agglutination tube tests for pregnancy are Pregnosticon Accuspheres.RTM. (Organon, Inc.) and Placentex.RTM. (Hoffman-LaRoche) which are latex tests and UCG-Lyphotest.RTM. (Wampole Labs) which is a hemagglutination test. These tube test kits have been evaluated by The Clinical Consumer Report, Vol. 2, No. 5 (March 1976) and found to be generally more accurate than slide agglutination tests in which the reagents and samples are combined on a microscope slide. There are also tube tests for pregnancy sold over-the-counter for home use. These are hemagglutination tests, i.e. Predictor.RTM. and Confidelle.RTM. (Denver Laboratories, Canada, Ltd.).
In any of these latex agglutination tests for pregnancy, whether direct or indirect, a change in the white suspension from a smooth colloidal state to a clumped, precipitated or agglutinated state must occur as an indicator of the presence (direct test) or absence (indirect test) of human chorionic gonadotropin (HCG). This distinction in white light may be difficult, particularly for the inexperienced technician as well as the untrained observer.
In the hemagglutination tube tests for pregnancy the indirect or inhibition method is generally used. In this test non-agglutinated HCG-coated red blood cells roll down the sides of the tube and settle in a ring pattern on the bottom indicating a positive test, whereas agglutinated cells form a loose network which stays on the sloping sides of the tube indicating a negative test. Some problems with this method are that the rings are not always well defined and if the tube is jarred during the incubation period the vibration may cause the loose network to break up and cells may slide down to the bottom of the tube resulting in inconclusive results characterized by atypical rings. See Am. J. Clin. Path., 64, 452 (1975) and Population Reports Series J, Number 7, pp. J-110-J-124 (1975) which is a publication of the Department of Medical and Public Affairs, The George Washington University Medical Center, Washington, D.C.
The present invention remedies the above mentioned problems in the prior art by making the outcome or test result of an immunological latex agglutination test much easier to discern and interpret. In this invention the observer has two indices of a test response which are (1) the appearance of a distinctly colored agglutant having formed from a smooth suspension of a contrasting color and (2) a color change in the test medium itself. Such a test will be advantageous not only to the experienced laboratory technician or physician but will also be suitable for use by the inexperienced individual in the home.
Another advantage of the present invention over hemagglutination inhibition tests is that the agglutant does not have to settle in a particular ring pattern. Thus, the test of the present invention is much less sensitive to accidental jarring or disturbances during the incubation period.